The field of diagnostics related to biochemical assays for the detection of chemicals, antigens and antibodies, biological species, metabolites, and related analytes has steadily advanced over the past decade. These advances are most notably in the area of medical diagnostics where a push towards more rapid and sensitive assays is the primary focus. As such, multiple novel technologies have blossomed including direct DNA and RNA detection, viral and bacterial identification, and novel reagents to assay for previously unknown biomarkers. Despite these advances, there still remains a gap between analyte detection and signaling of the detection event. Most assays still require a secondary step after binding of the analyte in order to signal that the event has occurred. This is commonly accomplished through the use of secondary reagents and visualization steps. Although these reagents are well known they do not fully address the need for an integrated detection and signaling method. As such, the present invention describes a method by which molecular recognition techniques may be coupled to existing dyes and signaling reagents in a fashion such that the signaling reagents have intrinsic detection and reporting capabilities.
The field of dye chemistry and in particular the use of dyes in chemical assays has been around for several centuries. In general, these dyes have been part of a multi-part chemical reaction or cascade whereby the dye responds indirectly to an upstream binding event. For example, in a typical glucose detection assay, glucose must first be oxidized by the enzyme glucose oxidase to yield gluconic acid and hydrogen peroxide. The byproduct hydrogen peroxide then reacts with a dye molecule to produce a visible color change. A typical glucose reaction involving the dye TMB (tetramethylbenzidine) which is oxidized by hydrogen peroxide in the presence of a peroxidase to induce a color change is shown:
Glucose OxidaseC6H12O6+O2→CH2(OH)(CHOH)4COOH+H2O2 
PeroxidaseH2O2+C16H20N2→color change+CO2 Additionally the high amounts of hydrogen peroxide produced by glucose require complimentary levels of dye which can lead to high optical density and quantitative difficulties.
A wide variety of naturally occurring and synthetically derived dyes are available and are used in applications ranging from textiles to simple colorants. Certain classes of dyes have been exploited in diagnostics due to their intrinsic ability to respond to particular conditions of their molecular environment such as pH, temperature, hydration, solvent polarity etc. A select group of these dyes in fact have the ability to bind small molecules and ions including cations such as Na, K, Cu, Zn, etc. Chrome Azurol S binds with Cu to form a blue complex, for example. Anions ranging from small organic molecules to halides such as fluoride may also be complexed. For example, alizarin complex one is known to complex fluoride. However, dyes with intrinsic capabilities to respond to complex analytes in general do not exist or are limited in their scope and/or use.
The field of molecular chemistry and in particular supra-molecular recognition is a relatively new addition to the established diagnostics community. Molecular recognition involves the rational design of complementary receptor complexes that are uniquely designed to bind a pre-determined analyte or chemical species. These receptors can be designed in a manner to bind a variety of analytes ranging from simple cations and anions to larger proteins, metabolites, chemical compounds, etc. Nevertheless, despite advances in molecular recognition, the use of a signaling reagent is still needed to visualize or report the binding/detection event.